Deliberately Losing Control of C−H Activation Processes in the Design of Small‐Molecule‐Fragment Arrays Targeting Peroxisomal Metabolism

Tareque, Raysa Khan; Hassell-Hart, Storm; Krojer, T.; Bradley, A.R.; Srikannathasan, Velupillai; Talon, R.; Fairhead, M.; Day, Iain J.; Bala, Kamlesh; Felix, Robert; Kemmitt, Paul D.; Brennan, P.E.; Delft, F. von; Saez, L. Diaz; Huber, K.; Spencer, John

doi:10.1002/cmdc.202000543

Cited by 4 publications

(4 citation statements)

References 41 publications

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“…extraction, solvent evaporation, purification, spectroscopic characterisation) were bypassed by the X-ray crystallographic analysis of the CRMs. 2,48 The workflow enabled the synthesis of a diverse >1000-member library of compounds in just 2 weeks, with analysis requiring a further 2 weeks, and yielding early structure activity relationships (SAR) derived from 3D structures, as well as compounds with on-scale biophysical affinity.…”

Section: Introductionmentioning

confidence: 99%

Efficient large-scale exploration of fragment hit progression by exploiting binding-site purification of actives (B-SPA) through combining multi-step array synthesis and HT crystallography.

Grosjean,

Aimon,

Hassell-Hart

et al. 2024

Preprint

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Fragment approaches are long-established in target-based ligand discovery. Nevertheless, their full transformative potential lies dormant, because progressing hits to potency remains difficult and underserved by methodology developments, which mostly focus on screening. The only credible progression paradigm is conventional design-make-test analyse (DMTA) medicinal chemistry, which is costly and thus necessitates picking winners early, thereby effectively discarding all the other hits. We here demonstrate the workability of an alternative strategy, namely immediate large-scale exploration of diverse hit-inspired compounds. The key insight is that it is effective to cheaply parallelize large numbers of non-uniform multi-step reactions, because even without compound purification, a high-quality readout of binding is available, namely crystallography of fragment screening. This has the sensitivity to detect even low-level binding of slightly active compounds, which the targeted binding site extracts directly from crude reaction mixtures (CRMs). In this proof-of-concept study, we expand a fragment hit from a crystal-based screen of the second bromodomain of human PHIP, using array synthesis on low-cost robotics to implement 6 independent multi-step reaction routes of up to 5 steps, attempting the synthesis of 1876 diverse expansions; designs were entirely driven by synthetic tractability. Expected product was present in 1108 CRMs, as detected by automated mass spectrometry; and 22 individual products were resolved in crystal structures of CRMs added to crystals. These provided an initial SAR map, revealed pose stability in 19 and instability in 3 products, and resolved stereochemical preference. Unexpectedly, in view of the naïve design approach, one resolved compound even showed on-scale biochemical potency (IC50=34 μM) and biophysical affinity (Kd=50 μM) after resynthesis. This binding-site purification of actives (B-SPA) process is formulaic and engineerable, here yielding the output of >25 person-years in ~20 days, with solvent use reduced from >4,500L to <20L. Thus, this approach, coupled with algorithmically guided compound and reaction design and new formalisms for data analysis, lends itself to routine fragment progression.

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Section: Introductionmentioning

confidence: 99%

Efficient large-scale exploration of fragment hit progression by exploiting binding-site purification of actives (B-SPA) through combining multi-step array synthesis and HT crystallography.

Grosjean,

Aimon,

Hassell-Hart

et al. 2024

Preprint

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show abstract

“…extraction, solvent evaporation, purification, spectroscopic characterisation) were bypassed by the X-ray crystallographic analysis of the crude reaction mixtures. [6,30] The workflow enabled the synthesis of a >1000-member library of compounds in a time frame of 2 weeks. The library was analysed within 2 weeks, allowing for rapid fragment expansion, based mainly on X-ray-derived crystallographic structure activity relationships (SAR).…”

Section: Introductionmentioning

confidence: 99%

High-throughput crystallography for rapid fragment growth from crude arrays by low-cost robotics

Grosjean

Aimon

Hassell-Hart

et al. 2023

Preprint

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We demonstrate that a simple workflow of array synthesis, combining low-cost robotics with analytic techniques to deconvolute crude reaction mixtures, is an effective way to collect structural data on a binding site. Starting from the high information content of the crystallographic fragment screens on PHIP(2) (second bromodomain of the pleckstrin homology domain interacting protein), a collection of more than 1800 compounds was enumerated. Several thousand Crude Reaction Mixtures (CRMs) were synthesized on one robotic platform, an OpenTrons OT-1 liquid handler, using reaction sequences of up to 5 chemical steps. Analysis via MScheck, an algorithm-based system for finding an m/z in a CRM, significantly shortened product identification protocol times. 969 usable X-ray diffraction datasets were acquired, which resolved as 22 reaction products binding to the protein, 19 with conserved poses relative to the original fragment and 3 with a new, unexpected binding pose. The 22 crystallographic hit compounds were subsequently tested with peptide displacement alpha-screen assay and time-resolved grating-coupled interferometry-based biosensor assays, which confirmed one molecule with an IC50 = 34 μM and KD = 50 μM, from an inactive fragment. The procedures described are entirely formulaic and engineerable and the method is eminently scalable. We anticipate that this cheap, low solvent-use approach will yield vast amounts of data, enabling rapid structural SAR landscape exploration around fragments, leading to faster fragment-to-lead times.

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“…Performing chemical reactions in an efficient manner, in terms of reduced solvent and energy use and higher yields, is desirable [1,2]. These include reactions that involve latestage functionalisation of key scaffolds or ones that "lose control" and produce a greater number of products for greater diversity for biological evaluation [3], e.g., in the synthesis of benzodiazepines or pyridine libraries [4][5][6][7]. Given that time is often a limiting factor, and a significant cost to factor in, processes that are "plug and play" and, can be carried out with little, or no, optimisation, are often de rigueur (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

A Convenient, Rapid, Conventional Heating Route to MIDA Boronates

et al. 2022

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Deliberately Losing Control of C−H Activation Processes in the Design of Small‐Molecule‐Fragment Arrays Targeting Peroxisomal Metabolism

Cited by 4 publications

References 41 publications

Efficient large-scale exploration of fragment hit progression by exploiting binding-site purification of actives (B-SPA) through combining multi-step array synthesis and HT crystallography.

Efficient large-scale exploration of fragment hit progression by exploiting binding-site purification of actives (B-SPA) through combining multi-step array synthesis and HT crystallography.

High-throughput crystallography for rapid fragment growth from crude arrays by low-cost robotics

A Convenient, Rapid, Conventional Heating Route to MIDA Boronates

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